The present invention pertains to a plasmid-meditated gene supplementation to alter pituitary development, and to increase prolactin levels, in an offspring of a female subject. More specifically, the present invention pertains to administering to a female subject a nucleic acid expression construct that encodes growth hormone releasing hormone (“GHRH”) to alter the pituitary development and pituitary hormone secretion (e.g. prolactin) in the offspring from the female subject.
The pituitary gland is an important link between the nervous system and the endocrine system. The pituitary gland is known to release many hormones that affect growth, sexual development, metabolism (e.g. protein, lipid and carbohydrate), glucocorticoids and the reproductive system. The pituitary gland has also been shown to release hormones that affect bone growth and regulate activity in other hormone secreting glands. This invention relates a method for altering pituitary gland development in offspring from female subjects that have been treated with a nucleic acid construct that encodes a growth hormone releasing hormone (“GHRH”) or functional biological equivalent. The expression of the GHRH or biological equivalent thereof is regulated by a tissue specific promoter (e.g. a myogenic promoter). When female subjects are treated with the nucleic acid construct that encodes GHRH, many physiological changes occur in the female subject directly. However, when female subjects are treated with the GHRH construct prior to, or during a gestation period, the offspring from these treated female subjects undergo similar physiological changes. For example, the subsequent expression and ensuing release of GHRH or biological equivalent thereof by the modified cells in the female subject results in the altered development of the pituitary gland in their offspring. Additionally, hormones secreted by the pituitary gland are increased in offspring from treated female subjects when compared to the offspring from control treated female subjects. More specifically, the pituitary gland is increased in sized and the levels of the multifunctional hormone prolactin is elevated utilizing this method.
The pituitary gland has two distinct parts, the anterior and the posterior lobes, each of which releases different hormones. The pituitary gland appears to be subservient in part to the hypothalamus. Pituitary gland development, including regulation and differentiation of somatotrophs, depends upon paracrine processes within the pituitary itself and involves several growth factors and neuropeptides. Secretion of growth hormone (“GH”) is stimulated by the natural GH secretagogue, called growth hormone releasing hormone (“GHRH”), and inhibited by somatostatin (“SS”). The central role of growth hormone (“GH”) is controlling somatic growth in humans and other vertebrates, and the physiologically relevant pathways that regulate GH secretion from the pituitary are well known. For example, the GH production pathway is composed of a series of interdependent genes whose products are required for normal growth. The GH pathway genes include: (1) ligands, such as GH and insulin-like growth factor-I (“IGF-I”); (2) transcription factors such as prophet of pit 1, or prop 1, and pit 1: (3) stimulatory and inhibitory factors, such as growth hormone releasing hormone (“GHRH”) and somatostatin (“SS”), respectively; and (4) receptors, such as GHRH receptor (“GHRH-R”) and the GH receptor (“GH-R”). These genes are expressed in different organs and tissues, including but not limited to the hypothalamus, pituitary, liver, and bone. Effective and regulated expression of the GH pathway is essential for optimal linear growth, as well as homeostasis of carbohydrate, protein, and fat metabolism. GH synthesis and secretion from the anterior pituitary is stimulated by GHRH and inhibited by somatostatin, which are both hypothalamic hormones. GH stimulates production of IGF-I, primarily in the liver, and other target organs. IGF-I and GH, in turn, feedback on the hypothalamus and pituitary to inhibit GHRH and GH release. GH elicits both direct and indirect actions on peripheral tissues, the indirect effects being mediated mainly by IGF-I.
The immune function is modulated by IGF-I, which has two major effects on B cell development: potentiation and maturation, and as a B-cell proliferation cofactor that works together with interlukin-7 (“IL-7”). These activities were identified through the use of anti-IGF-I antibodies, antisense sequences to IGF-I, and the use of recombinant IGF-I to substitute for the activity. There is evidence that macrophages are a rich source of IGF-I. The treatment of mice with recombinant IGF-I confirmed these observations as it increased the number of pre-B and mature B cells in bone marrow. The mature B cell remained sensitive to IGF-I as immunoglobulin production was also stimulated by IGF-I in vitro and in vivo.
The production of recombinant proteins in the last 2 decades provided a useful tool for the treatment of many diverse conditions. For example, recombinant GH administration has been used to treat GH-deficiencies in short stature children, or as an anabolic agent in burn, sepsis, and as well as in the elderly and AIDS patients. However, resistance to GH action has been reported in malnutrition and infection. Long-term studies on transgenic animals and in patients undergoing GH therapies have shown no causal correlation between GH or IGF-I therapy and cancer development. GH replacement therapy is widely used clinically, with beneficial effects, but therapy is associated several disadvantages: GH must be administered subcutaneously or intramuscularly once a day to three times a week for months, or usually years; insulin resistance and impaired glucose tolerance can occur; accelerated bone epiphysis growth and closure has been observed in pediatric patients (Blethen, S. L., et al. 1996).
In contrast, essentially no side effects have been reported for recombinant GHRH therapies. Extracranially secreted GHRH, as mature peptide or truncated molecules (as seen with pancreatic islet cell tumors and variously located carcinoids) are often biologically active and can even produce acromegaly (Esch, et al., 1982; Thorner, et al., 1984). Administration of recombinant GHRH to GH-deficient children or adult humans augments IGF-I levels, increases GH secretion proportionally to the GHRH dose, yet still invokes a response to bolus doses of recombinant GHRH (Bercu and Walker, 1997). Thus, GHRH administration represents a more physiological alternative of increasing subnormal GH and IGF-I levels (Corpas, et al., 1993).
GH is released in a distinctive pulsatile pattern that has profound importance for its biological activity (Argente, et al., 1996). Secretion of GH is stimulated by the GHRH, and inhibited by somatostatin, and both are hypothalamic hormones (Thorner, et al., 1995). GH pulses are a result of GHRH secretion that is associated with a diminution or withdrawal of somatostatin secretion. In addition, the pulse generator mechanism is timed by GH-negative feedback. The endogenous rhythm of GH secretion becomes entrained to the imposed rhythm of exogenous GH administration. Effective and regulated expression of the GH and insulin-like growth factor-I (“IGF-I”) pathway is essential for optimal linear growth, homeostasis of carbohydrate, protein, and fat metabolism, and for providing a positive nitrogen balance (Murray, et al., 2000). Numerous studies in humans, sheep or pigs showed that continuous infusion with recombinant GHRH protein restores the normal GH pattern without desensitizing GHRH receptors or depleting GH supplies as this system is capable of feed-back regulation, which is abolished in the GH therapies (Dubreuil, et al., 1990). Although recombinant GHRH protein therapy entrains and stimulates normal cyclical GH secretion with virtually no side effects, the short half-life of GHRH in vivo requires frequent (one to three times a day) intravenous, subcutaneous or intranasal (requiring 300-fold higher dose) administration. Thus, as a chronic treatment, recombinant GHRH administration is not practical.
Wild type GHRH has a relatively short half-life in the circulatory system, both in humans (Frohman, et al., 1984) and in farm animals. After 60 minutes of incubation in plasma, 95% of the GHRH(1-44)NH2 is degraded, while incubation of the shorter (1-40)OH form of the hormone, under similar conditions, shows only a 77% degradation of the peptide after 60 minutes of incubation (Frohman, et al., 1989). Incorporation of cDNA coding for a particular protease-resistant GHRH analog in a gene transfer vector results in a molecule with a longer half-life in serum, increased potency, and provides greater GH release in plasmid-injected animals (Draghia-Akli, et al., 1999, herein incorporated by reference). Mutagenesis via amino acid replacement of protease sensitive amino acids prolongs the serum half-life of the GHRH molecule. Furthermore, the enhancement of biological activity of GHRH is achieved by using super-active analogs that may increase its binding affinity to specific receptors (Draghia-Akli, et al, 1999).
Extracranially secreted GHRH, as processed protein species GHRH(1-40) hydroxy or GHRH(1-44) amide or even as shorter truncated molecules, are biological active (Thorner, et al., 1984). It has been reported that a low level of GHRH (100 pg/ml) in the blood supply stimulates GH secretion (Corpas, et al., 1993). Direct plasmid DNA gene transfer is currently the basis of many emerging gene therapy strategies and thus does not require viral genes or lipid particles (Muramatsu, et al., 1998; Aihara and Miyazaki, 1998). Skeletal muscle is target tissue, because muscle fiber has a long life span and can be transduced by circular DNA plasmids that express over months or years in an immunocompetent host (Davis, et al., 1993; Tripathy, et al., 1996). Previous reports demonstrated that human GHRH cDNA could be delivered to muscle by an injectable myogenic expression vector in mice where it transiently stimulated GH secretion to a modes extent over a period of two weeks (Draghia-Akli, et al., 1997).
Administering novel GHRH analog proteins (U.S. Pat. Nos. 5,847,066; 5,846,936; 5,792,747; 5,776,901; 5,696,089; 5,486,505; 5,137,872; 5,084,442, 5,036,045; 5,023,322; 4,839,344; 4,410,512, RE33,699) or synthetic or naturally occurring peptide fragments of GHRH (U.S. Pat. Nos. 4,833,166; 4,228,158; 4,228,156; 4,226,857; 4,224,316; 4,223,021; 4,223,020; 4,223, 019) for the purpose of increasing release of growth hormone have been reported. A GHRH analog containing the following mutations have been reported (U.S. Pat. No. 5,846,936): Tyr at position 1 to His; Ala at position 2 to Val, Leu, or others; Asn at position 8 to Gln, Ser, or Thr; Gly at position 15 to Ala or Leu; Met at position 27 to Nle or Leu; and Ser at position 28 to Asn. The GHRH analog is the subject of U.S. patent application Ser. No. 09/624,268 (“the '268 application”), which teaches application of a GHRH analog containing mutations that improve the ability to elicit the release of growth hormone. In addition, the '268 application relates to the treatment of growth deficiencies; the improvement of growth performance; the stimulation of production of growth hormone in an animal at a greater level than that associated with normal growth; and the enhancement of growth utilizing the administration of growth hormone releasing hormone analog and is herein incorporated by reference.
U.S. Pat. No. 5,061,690 is directed toward increasing both birth weight and milk production by supplying to pregnant female mammals an effective amount of human GHRH or one of it analogs for 10-20 days. Application of the analogs lasts only throughout the lactation period. However, multiple administrations are needed. A co-pending disclosure regarding administration of the growth hormone releasing hormone (or factor) as a DNA molecule, such as with plasmid mediated therapy techniques has been disclosed (U.S. patent application Ser. No. 10/021,403).
U.S. Pat. No. 5,134,120 (“the '120 patent”) and U.S. Pat. No. 5,292,721 (“the '721 patent”) teach that by deliberately increasing growth hormone in swine during the last 2 weeks of pregnancy through a 3 week lactation resulted in the newborn piglets having marked enhancement of the ability to maintain plasma concentrations of glucose and free fatty acids when fasted after birth. In addition, the '120 and '721 patents teach that treatment of the sow during lactation results in increased milk fat in the colostrum and an increased milk yield. These effects are important in enhancing survivability of newborn pigs and weight gain prior to weaning. However, the '120 and '721 patents provide no teachings regarding administration of the growth hormone releasing hormone (“GHRH”) as a DNA form.
Prolactin is a single-chain protein hormone closely related to growth hormone. It is chiefly secreted by lactotrophs in the anterior pituitary. However, prolactin is also synthesized and secreted by a broad range of other cells in the body, most prominently various immune cells, the brain and the decidua of the pregnant uterus. Prolactin is also found in the serum of normal females and males. Prolactin secretion is pulsatile and also shows diurnal variation, with the serum concentration increasing during sleep and the lowest level occurs about 3 hours after waking. The secretion of prolactin is increased by stress and appears to be dependent upon a women's estrogen status.
The conventional view of prolactin is that the mammary gland is its major target organ, and stimulating mammary gland development along with milk production define its major functions. Although these views are true, such descriptions fail to convey an accurate depiction of this multifunctional hormone. For example, it is difficult to find a mammalian tissue that does not express prolactin receptors, and although the anterior pituitary is the major source of prolactin, the hormone is synthesized and secreted in many other tissues. Overall, several hundred different actions have been reported for prolactin in various species. Some of prolactin's major effects are summarized below.
Prolactin's major known functions are attributed with mammary gland development, milk production and reproduction. In the 1920's it was found that extracts of the pituitary gland, when injected into virgin rabbits, induced milk production. Subsequent research demonstrated that prolactin has two major roles in milk production:                Prolactin induces lobulo-alveolar growth of the mammary gland, wherein the alveoli are the clusters of cells in the mammary gland that actually secrete milk.        Prolactin stimulates lactogenesis or milk production after giving birth. Prolactin, along with cortisol and insulin, act together to stimulate transcription of the genes that encode milk proteins. The critical role of prolactin in lactation has been established by utilizing transgenic mice with targeted deletions in the prolactin gene. Female mice that are heterozygous for the deleted prolactin gene only produce about half the normal amount of prolactin, and fail to lactate after their first pregnancy.        
Prolactin is also important in several non-lactational aspects of reproduction. For example, in some species (e.g. rodents, dogs, skunks), prolactin is necessary for maintenance of ovarian structures (i.e. corpora lutea) that secrete progesterone. Mice that are homozygous for an inactivated prolactin gene and thus incapable of secreting prolactin are infertile due to defects in ovulation, fertilization, preimplantation development and implantation. Prolactin also appears to have stimulatory effects in some species on reproductive or maternal behaviors such as nest building and retrieval of scattered young.
Prolactin also appears to elicit effects in the immune system. For example, the prolactin receptor is widely expressed by immune cells, and some types of lymphocytes synthesize and secrete prolactin. These observations suggest that prolactin may act as an autocrine or paracrine modulator of immune activity. Conversely, mice with homozygous deletions of the prolactin gene fail to show significant abnormalities in immune responses. A considerable amount of research is in progress to delineate the role of prolactin in normal and pathologic immune responses. However, the significance of these potential functions remains poorly understood.
Administering prolactin stimulating hormones, or prolactin agonists (U.S. Pat. Nos. 5,605,885; and 5,872,127) for the purpose of stimulating the immune system have been reported. The U.S. Pat. No. 5,872,127 (“the '127 patent”) filed by Cincotta in 1999 discloses methods for treating a disorder of the immune system or an immunodeficiency state that comprise the steps of administering to a patient an effective amount a serotonin agonist and at a dopamine agonist, where the combination of the serotonin agonist and the dopamine agonist are present in an amount effective to treat a patient's immuno-compromised condition. The administration of each of the agents is confined to a specific time of day that is capable of adjusting the prolactin profile of the patient to conform or to approach the standard human prolactin profile.
Additionally, the supplementation of the prolactin agonists in U.S. Pat. No. 5,605,885 (“the '885 patent”) disclose a method for the stimulation of a suppressed or deficient immune system by regulating the blood levels or activity of the hormone prolactin directly. The '885 patent method comprises treating an immunosuppressed subject with proteins, peptides and compounds that have prolactin-like activity including, but not limited to, prolactin, peptide sequences from prolactin that have prolactin-like activity, growth hormone (a structurally similar and biologically related hormone), or peptide sequences from growth hormone which have prolactin-like activity, placental lactogens, and any genetically engineered protein sequence which has prolactin-like activity. However, neither the '885 and '127 patents provide teachings regarding increasing prolactin levels by the administration of the growth hormone releasing hormone (“GHRH”) as a DNA form.
In summary, the production of recombinant proteins in the last 2 decades provides a useful tool for the treatment of many diverse conditions, however these treatments have some significant drawbacks. It has also been demonstrated that nucleic acid expression constructs that encode recombinant proteins are viable solutions to the problems of frequent injections and high cost of traditional recombinant therapy. By utilizing knowledge of specific pituitary/hypothalamic pathways and the functionality of extracranially secreted hormones, it is possible to treat many conditions utilizing a plasmid-mediated introduction of a nucleic acid construct into a subject. Furthermore, it has been shown that some beneficial effects can be conferred to the offspring of female subjects that have been treated utilizing recombinant proteins during gestation and without treating the offspring directly. Thus, this invention is related to the conferred beneficial effects in offspring from GHRH treated mothers. More specifically this invention discloses methods for altering pituitary development and pituitary hormone secretion (e.g. prolactin) in the offspring from female subjects treated with nucleic acid constructs that encode GHRH.